Accurate measurement of enzyme kinetics is an essential part of understanding the mechanisms of biochemical reactions. The typical means of studying such systems use stirred cuvettes, stopped-flow apparatus, microfluidic systems, or other small sample containers. These reaction-kinetics measurements reactors are usually made of quartz, glass, or an easily-moldable polymer such as polydimethylsiloxane. For studying enzyme kinetics or free radical chemistry, using small volumes with high concentrations is desirable, but interactions of biochemical moieties with these materials can be problematic. The main obstacle is that, as the system volume shrinks, the ratio of surface area to volume increases. As a result, the importance of adsorption can increase, and proteins sticking to walls, biofilms grow on walls, and radicals annihilating on walls can become significant. Thus, under these circumstances, typical microfluidic systems are incompatible with the demands of accurate study of many biochemical systems.
Over the past five years, acoustic levitation has become a technique uniquely suited to studying biochemical reactions. Acoustic levitation offers several advantages over typical reaction systems, including small sample volume (and mass), the prevention of chemical contamination between drops and external objects, rapid mixing within the drop as a result of circulation driven by the levitating ultrasound, and freedom from wall interactions. We have developed an Acoustically-Levitated Drop Reactor (LDR) to study enzyme-catalyzed reaction kinetics related to free-radical and oxidative stress chemistry.
Microliter-scale droplet generation, reactant introduction, maintenance, and fluid removal are all important aspects in conducting reactions in a levitated drop. Therefore, we developed a three-capillary bundle system to address these needs. Since the capillary system is used to introduce drops to the levitation cavity, our LDR system is not 100% wall-less, but the ratio of solid surface area to drops surface area is less than 20%, which is low compared to all the other reaction systems. The largest interface seen by reactants is a liquid/gas interface.
Herein, I report kinetic measurements for both luminol chemiluminescence and the reaction of pyruvate with nicotinamide adenine dinucleotide, catalyzed by lactate dehydrogenase in a levitated drop. Observations of the chemiluminescence experiment showed second-order kinetics were detected within experimental error prior to deviations seen after 10 s. For the enzyme-catalyzed reaction kinetic measurements, it was found that the KM for lactate dehydrogenase matched literature values to better than one standard deviation of the literature experiment. Also studied were the effects of laser exposure on the stability of Myeloperoxidase at the liquid/gas interface. It was found that although myeloperoxidase degrades over several hours in buffer, there is no indication that the degradation is due to either denaturation at the free surface or to two-photon photolysis.
Based on our accomplishments, we demonstrate the feasibility of using a levitated drop as a microreactor. The reactions discussed utilized in-house developed fiber optic and laser-based optical detection systems to monitor reaction kinetics inside a levitated drop via chemiluminescence and fluorescence. Outgrowths of these accomplishments will hopefully lead to the use of additional detection systems (including electrochemical and mass spectrometry) with the LDR to study a wide variety of biochemical reaction kinetics.